Tumor and Metastatization
Tumorigenesis is a multi-stage process in which some cells progressively evolve toward malignity. The actual knowledge in the field of neoplasia underlines that cancer is a disease induced by dynamic changes of the genome. Through these variations, tumor cells acquire independence from various mechanisms that control the physiological functions of the organism. As a consequence, they become able of (1) growing continuously, (2) inducing the recruitment of endothelial cells for the formation of new blood vessels and (3) colonizing organs different from that of origin.
The proliferative capability of tumor cells is fundamentally due to two mechanisms. First, while normal cells need mitogenic factors to switch from a quiescent condition to an active, proliferating one, in tumor cells mutations and/or overexpression of growth factor receptors can induce the proliferation cascade independently from the presence of a ligand. Moreover, in many cases, tumor cells acquire the ability to synthesize soluble factors they are sensitive to. So, an autocrine stimulation is set up, which further enhances the tumor growth. The other phenomenon of deregulation of the cell growth in tumors is the resistance to apoptosis (programmed cell death). This mechanism, fundamental in the growth and remodeling of organs during the physiological development, is induced also in the case of non-reversible genome damages, to avoid the expansion of aberrant cell population. Some cells, however, can escape this kind of protection and become independent, thus favoring the propagation of mutations and the consequent neoplastic progression.
A primordial tumor mass is constituted by a small cell number, and could not develop over 2 mm in diameter if not sustained by adequate feed and oxygen support. This is the phase in which angiogenesis, the formation of new blood capillaries from pre-existing blood vessels, is strongly stimulated by tumor cells themselves. The unbalance between positive and negative signals of the angiogenesis regulation leads to the neo-formation of a vascular network that penetrates and feeds the actively proliferating tumor mass. Tumor blood vessels, besides providing the nutrition, have the function of carrying malignant cells toward other body districts.
Tumor progression evolves toward an irreversibility stage, whose characteristic feature is metastatization. In this process, pioneer cells escape from the primary tumor mass. Once entered into the capillaries that fill the tumor, they reach the bloodstream, which carries them in regions distant from the site of derivation, where they will give raise to a secondary tumor.
The development of metastasis represents a complex biological event, related to the interactions between intrinsic factors of the organism (general conditions, integrity of the immune response) and specific features of tumor cells (localization, size and histological patterns). From the microscopic primary site, the diffusion of tumor cells is first local, through a centrifugal spreading. By producing proteases that degrade the intercellular connections and the extracellular matrix, tumor cells invade anatomic structures and tissues that are scarcely resistant (fat tissue, nerve sheaths, bone marrow).
A first obstacle to the metastatic diffusion is offered by the presence of relatively impenetrable structures, such as the organ capsules, cartilage or periostium, the meninx. Due to the difficulty of going beyond these boundaries, metastatization in distant sites must follow steps that can be summarized as follows: (1) entrance into the tumor capillary network by a mechanism called “intravasation”, (2) transport through the bloodstream, (3) specific recognition of the destination endothelium, (4) exit from the capillary by a mechanism called “extravasation” and (5) metastasis development, supported by active angiogenesis.
The success of dissemination depends on the anatomical features and on the hemodynamic factors of the host organism, and on the interactions that tumor cells undergo with the endothelium lining the blood vessels. The most common pathways of diffusion are the vessels (lymphatic and blood) and the celomatic cavities. Lymphatic vessels are quite easily penetrated, because of the absence of a basal lamina. So, tumor cells can easily transit into the lymph nodes, before entering the venous system through the lymphatic-venous connections. The transport into the vessels can affect both arterious and venous system, even if the venous invasion is more common, because the venous circulation collects the flux exiting from organs. Typical examples are the systemic vein for the lung or the port vein for the liver. The trans-celomatic dissemination instead concerns the pleural cavity of the chest and the peritoneal spaces of the abdomen and pelvis. The most commonly involved site is peritoneum, where, after pouring liquids due to the obstruction of the hepatic veins, tumor cells are collected in the ascitic fluid. Stomach, colon, pancreas and ovary cancers usually take this system.
Tumor metastatic cells express specific molecular determinants that contribute in various ways to the metastasis itself. The distribution of metastasis is not casual, but each tumor has preferential addresses, this is known as organo-tropism. Liver is a target organ for colorectal tumors; bones for prostate and ovary tumors; lungs for testis, bone and breast tumors. Lungs and liver, due to their filter function and to the presence of a huge number of capillaries, can receive metastases virtually from every organ and also send tumor colonies, mainly toward brain and bones.
The liver is a common site for metastatic lesions. The reason has to be searched in the functional and structural organization of the hepatic district. The port vein, which drains the blood to the abdominal viscera, represents the conduct through which the cells coming from the primary tumors are veiculated to the liver. The adhesion of circulating tumor cells to liver endothelium is a critical step for the beginning of metastatization. Hepatic metastases develop as a consequence of the invasion of the hepatic parenchyma by these cell thrombi.
The high volume of hepatic blood flux (about 25% of the cardiac flux), and the particular microscopic anatomy of the sinusoids are the factors that favor the hepatic dissemination. The primary tumor may be localized in the gastro-intestinal tract, i.e. colon, rectum, stomach, pancreas, biliary tract and bowel. To those, also tumors of the breast and lung may be added.
Colorectal Tumor
Different kinds of classification exist that, in general, divide the progressive evolution of the disease in steps characterized by the degree of body invasion of that tumor. The Dukes and MAC (Modified Astler-Coller) classifications, proposed at the beginning of the clinical studies, are now the less used. Generally, the TNM (Tumor Node Metastasis) classification is preferred, which includes four successive stages:                stage I: tumor limited to the mucosa and the sub-mucosa;        stage II: extension to deeper layers of the intestinal wall;        stage III: invasion of sub-sierosa and lymph nodes;        stage IV: metastasis.        
The therapeutic approaches more common by now are surgery, chemotherapy and radiotherapy. The kind of clinical strategy is chosen based on the stage in which the pathology is. In general, the following protocols are used:                stage I: surgery (colostomy);        stage II: surgery can be associated to chemotherapy;        stage III: surgery is in any case associated with chemotherapy;        stage IV: palliative treatment with surgery and/or chemotherapy.        
Liver is the most frequent site of colonization by primary colorectal cancer. Currently, the only treatment with a curative potential is surgical removal of metastases. However, despite the increasingly effective means of the hepatic surgery, most patients with liver metastases are not amenable for surgery, because of the extension of their tumor mass.
A Different Approach to Cancer Therapy: Attacking Tumor Blood Vessels
The chemotherapic drugs currently used are between the drugs with the most narrow therapeutic window in the whole medical field. As a consequence, the dose of antitumor drugs that can be administered is limited by the toxic effects on normal tissues. This difficulty can be overcome by targeting cytotoxic drugs to the tumor itself. Even if this has been a goal for long time in cancer biology and in oncological medicine, right now only few examples are known in which the target administration of a drug is possible. For example, the use of monoclonal antibodies against tumor antigens had a limited success, since only a few tumor antigens are known and generally antibodies poorly penetrate into tissues. Moreover, since tumor cells are genetically instable and growth-advantageous mutations accumulate, tumor cell-targeted treatments are generally followed by clonal selection of resistant cells.
The targeting of therapy to the tumor vascular network allows to overcome some of the problems related to traditional therapy. Endothelial cells in the tumor vascular system express molecules peculiar of anogiogenic vessels. Vascular targeting offers several advantages. First, endothelial lining is easily accessible. On the contrary, a tumor-targeted drug needs to diffuse on long distances, penetrate into tightly bound tumor cells and in a very dense stroma, and contrast a very high interstitial pressure. Second, since tumor cells depend on blood supply for their growth, a tumor therapy addressed to the vessels does not need to lead to the destruction of all the endothelial cells. Indeed, endothelium-target therapy has an intrinsic amplification mechanism. Finally, since endothelial cells are diploid and not transformed, it is improbable that they loose the expression of a surface receptor or acquire drug resistance through mutations and clonal evolution. Some endothelial markers have been recently identified. Among these molecules there are some integrins, particularly αvβ3 and αvβ5 and endothelial tyrosine kinase receptors with their cognate ligands (VEGF receptors and the various VEGFs, Tie1, Tie2 and angiopietins).
Peptides that Target a Mouse Model of Human Tumor: Discovery of Tumor Endothelial Markers
By phage display studies performed in vivo in different animal models peptide sequences have been identified which are able to selectively target tumor vascularization. These sequences proved to be a valid instrument to characterize tumor endothelium and its specific molecular determinants, and to develop biotechnological applications in tumor therapy.
In this way, recurrent peptide sequences have been identified, such as RGD (Arginin-Glycin-Aspartic acid) and NGR (Asparagin-Glycin-Arginin). The RGD motif is embedded in the sequence of several proteins of the extracellular matrix and represents their interaction site with integrins. A phage that presents the CDRGDCFC (SEQ ID NO: 203) sequence, named RGD-4C, is able to specifically target breast tumors, and to selectively bind the αvβ3 and αvβ5 integrins. In vitro experiments demonstrated that RGD-containing peptides inhibit cell-cell adhesion thus inducing apoptosis. So, it has been thought that the RGD peptide, without further modification, can act as an antiangiogenic drug, leading to cell death after disruption of the cell-matrix interactions. Also the NGR peptide binds integrins, even if with minor affinity compared to RGD. The specific receptor for the NGR sequence has been successively identified in another membrane protein, aminopeptidase N (APN), overexpressed in vascular structures in active angiogenesis and not detectable in quiescent endothelium. It has been demonstrated that APN specific antibodies can inhibit retinal neovascularization induced by hypoxia in the mouse. In the same way, mice treated with anti-APN antibodies have breast tumors strongly regressed compared to the control group.
In another set of studies peptides that specifically bind the NG2 proteoglycan have been identified, a mouse homolog of HMP (human melanoma proteoglycan), also known as Molecular Weight Melanoma-Associated Antigen. This proteoglycan is mainly expressed by glial progenitor cells, skeletal muscle and cartilage. After the differentiation, the NG2 surface expression is lost. In adults, its presence is limited to vessels in active angiogenesis in some tumor kinds, among which glioblastoma, condrosarcoma, melanoma, and some leukemias. In a nude mice bearing a malignant melanoma, an anti-NG2 antibody conjugated with doxorubicin suppresses tumor growth.
Peptides as Antitumor Drugs
Remodeling of the extracellular matrix is common both to endothelial activation and neoplastic invasion, and need the action of particular enzymes called Matrix Metallo-Proteases (MMP). These proteases, overexpressed in the tumor, are almost absent in normal tissues, except in cell migration and tissue remodeling events during morphogenesis. Synthetic inhibitors of two such proteases, MMP-2 (Gelatinase A; 72 Kd) and MMP-9 (Gelatinase B; 92 Kd), which are the more strictly involved proteases in angiogenesis and metastatic potential, have been isolated by phage display. From this study, it has been shown that the most represented clones express the LRSGRG (SEQ ID NO: 204) sequence derived from a CX6C library. Another protein family, identified from a CX9 collection, is the one with the HWGF (SEQ ID NO: 205) motif. Soluble peptides containing the HWGF (SEQ ID NO: 205) motif show in vitro inhibitory activity against MMP-9. These peptides inhibit the migration of tumor cell lines and of endothelial cells derived from human umbilical cord. In vivo, they are efficient in inhibiting tumor growth and in preventing the appearance of metastases.
Use of Peptides in Biotechnologically Innovative Antitumor Therapies
As described previously, peptides specifically associated to tumor endothelial markers or tumor cells have been successfully employed in therapeutic protocols in the mouse. A second approach has been investigated, conjugating RGD-4C and CNGRC (SEQ ID NO: 207) peptides to the chemotherapic drug doxorubicin, and using this compound for the treatment of breast tumors in mice. Animals subjected to this therapy survived up to six months, demonstrating that this compound is able to inhibit both primary tumor and metastasis development with higher efficacy and lower toxicity compared to systemic administration.
In a third set of applications, chimeric peptides have been made, which possess two functional domains. The former can selectively bind to the target cell and be internalized; the latter is pro-apoptotic, non toxic in body fluids but only in the intracellular environment. More than 100 peptides exist that act causing the destruction of mitochondrial membranes and induction of apoptosis. Among these, a 14 aa sequence has been selected, KLAKLAKKLAKLAK (SEQ ID NO: 206), which demonstrated to have a strong antibiotic potential in the form of D-enantiomer. The peptides RGD-4C and CNGRC (SEQ ID NO: 207) have been coupled to this peptide. It has been found that these compounds cause mitochondrial alterations and lead to morphological variations typical of an apoptotic status, such as condensation and fragmentation of the nuclear structures. These results have been confirmed in vivo: mice to which the antitumor agent has been administered show tumors of reduced size and survive for several months.